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[1]
mechanisms . Depending on such factors as cellular origin, cargo contents, membrane composition,
and target cell indications, interactions of EV with damaged, diseased, or otherwise compromised tissue
[1,2]
beds can promote functional recovery . As membrane-bound vesicles, EV are typically divided into two
groups based on size and mode of formation. Microvesicles (MV) tend to be larger (100 nm to 1 µm), and
[3]
are directly assembled from cellular contents and released by outward budding of the cell membrane .
Exosomes are smaller (30-100 nm) intraluminal vesicles within endosome-derived multivesicular bodies
[4]
that then fuse with and release from the plasma membrane . For the resolution of radiation injury, no
clear evidence has demonstrated a therapeutic advantage of human stem cell-derived MV over exosomes or
vice versa, so the EV-based treatments used in this study include the full-size range of vesicles secreted into
the conditioned medium by the proliferating human neural stem cells.

Compared to stem cell therapies, the ability of EV to stimulate regenerative healing while eliminating
risks of teratoma/tumor formation and confounding complications associated with immune suppression,
indicate their potential translational utility. While regenerative approaches for implementing stem cell
[5]
treatments in the context of radiation injury hold tremendous potential , EV circumvents certain stem
cell-based caveats due to their low immunogenicity, long-circulating half-life and ability to cross the blood-
brain barrier [2,6,7] . Recent work from our laboratory has demonstrated the functional equivalence of EV
[8,9]
and human stem cells following intra-cranial delivery to the irradiated hippocampus . These studies
demonstrated that both therapies mitigated radiation-induced cognitive dysfunction while preserving host
neuronal morphology and attenuating neuroinflammation . EV-based therapies have also been used to
[8,9]
[10]
reduce indications of neuroinflammation or cognitive dysfunction associated with chemobrain , other
neurodegenerative conditions [11,12] , and physical injury [13-15] , indicating their widespread tolerance and broad
efficacy in the brain.

Migration of EV through the extracellular space or circulation provides the routes whereby EV can
interact with target cells, presumably through interactions between transmembrane proteins on the EV
and specific receptors on the surface of the target cell. Recipient cells internalize EV via either fusion with
the plasma membrane or more commonly by endocytosis . This then initiates the functional transfer of
[16]
critical bioactive cargo containing lipids, proteins, organelles, and an assortment of nucleic acids including
microRNA (miRNA). The ability of EV to target and functionally interact within the radiation-injured
tissue bed provides a heretofore unexplored area for resolving a wide range of dose-limiting normal tissue
toxicities associated with the radiotherapeutic management of cancer. For these reasons we embarked on a
targeted technical study to evaluate whether other non-surgical administration routes could deliver hNSC-
derived EV to the parenchyma of the brain.

METHODS
Stem cell culture, isolation, and labeling of EV
Growth, culturing and maintenance of human neural stem cells (hNSC, H9-derived ENstem-A, Millipore)
was approved by the Institutional Human Stem Cell Research Oversight (HSCRO, #2007-5629) and
Institutional Biosafety Committees. The growth of hNSC and harvest of conditioned medium from hNSC
has been described previously [9,10] . All cultures were tested and found to be negative for mycoplasma with
MycoAlert Mycoplasma Detection Kit (Lonza, Cat# LT07-118; Basel, Switzerland).

For in vivo tracking, EV were labeled with PKH26 (Sigma-Aldrich, Cat# PKH26PCL; St. Louis, MO)
the day before transplantation. The EV were then resuspended in Diluent C and incubated with Dye
Solution for 2 min with intermittent mixing as per the manufacturer’s protocol. The dye was quenched
with 1% bovine serum albumin in water, and EV were isolated through ultracentrifugation and washed as
[17]
described .